Abstract:

The invention relates to a method for the diagnosis and/or risk
stratification of cardiac insufficiency, according to which the marker
neurophysin or a fragment or partial peptide thereof is determined on
patients to be examined. The invention also relates to advantageous
marker combinations containing neurophysin, and to a diagnostic device
and a kit for carrying out the method.

Claims:

1. Method for the diagnosis and/or risk stratification of cardiac
insufficiency, characterized in that a determination of the marker
neurophysin or a fragment or partial peptide thereof is carried out on a
patient to be examined.

2. Method according to claim 1, characterized in that the method is an
in-vitro diagnosis.

4. Method for the diagnosis and/or risk stratification of cardiac
insufficiency according to claim 1 for carrying out clinical decisions,
in particular further treatment and therapy by means of pharmaceuticals,
in particular in intensive care medicine or emergency medicine and for
the hospitalization of the patient.

5. Method for the diagnosis of cardiac insufficiency according to claim 1
for the prognosis, for differential diagnostic early detection and
detection, for the assessment of the degree of severity and for the
assessment of the course of a cardiac insufficiency accompanying therapy.

6. Method according to claim 1, characterized in that in addition a
determination is carried out on a patient to be examined with at least
one further marker selected from the group of inflammatory markers,
cardiovascular markers, neurohormonal markers or ischemic markers.

7. Method according to claim 1, characterized in that the inflammatory
marker is selected from at least one marker from the group of C-reactive
protein (CRP), cytokinin, such as, for example, TNF-alpha, interleukins,
such as, for example, IL-6, procalcitonin (1-116, 3-116) and adhesins,
such as VCAM or ICAM.

9. Method according to claim 1, characterized in that the ischemic marker
is selected from at least one marker from the group troponin I and T,
CK-MB.

10. Method according to claim 1, characterized in that the neurohormonal
marker is at least one natriuretic protein, in particular ANP (or ANF),
proANP, NT-proANP, BNP, proBNP, NT-proBNP or a partial sequence thereof.

11. Method according to claim 1, characterized in that parallel or
simultaneous determinations of the markers are carried out.

12. Method according to claim 1, characterized in that the determinations
are carried out on at least one patient sample.

13. Method according to claim 1, characterized in that the determinations
are carried out on an automatic analyzer, in particular by means of a
Kryptor.

14. Method according to claim 1, characterized in that the determinations
are carried out by means of a rapid test, in particular in
single-parameter or multiple-parameter determinations.

15. (canceled)

16. Diagnostic for carrying out a method according to claim 1.

17. Kit for the diagnosis and/or risk stratification of cardiac
insufficiency, containing analytical reagents for determining the marker
neurophysin or a fragment or partial peptide thereof and optionally
further markers according to claim 6 and auxiliaries.

Description:

[0001]The invention relates to a method for the diagnosis and/or risk
stratification of cardiac insufficiency, wherein a determination of the
marker neurophysin or a fragment or partial peptide thereof is carried
out on a patient to be examined. The invention relates to advantageous
marker combinations containing neurophysin. Furthermore, the invention
relates to a diagnostic device and a kit for carrying out the method.

[0003]In the early stages the patient often notices the cardiac
insufficiency only a little. Untreated, the disease as a rule increases
in severity and in the late stage leads to complete physical exhaustion
even at rest. The lack of nourishment of all of the body's organs,
including the cardiac itself can lead to death at this stage. Once the
disease is advanced, life expectancy is considerably reduced even with
optimum therapy (approx. 30% of deaths per year). It is therefore
important to recognize a cardiac insufficiency as early as possible and
to deal with its causes consistently.

[0004]In order to start a suitable therapy, an early diagnosis and
differentiation of the underlying disease is therefore necessary at an
early stage and in emergency and intensive medicine. Both the
differentiation and delimitation of cardiac insufficiency from other
diseases are often made difficult by unspecific symptoms (respiratory
distress, coughing).

[0005]A test is available using the determination of the plasma
concentration of the brain natriuretic peptide (BNP or NTproBNP), which
is also used in the course of daily routine for the diagnosis of cardiac
insufficiency (Maisel et al. (supra)).

[0007]In the prior art there is a great need to identify and provide
further markers for the indication of cardiac insufficiency and to supply
them to clinical practice.

[0008]It is therefore the object of the present invention to provide a
method for the diagnosis and/or risk stratification of cardiac
insufficiency.

[0009]However, the disadvantage of known diagnostic methods using the
markers previously known is that an early and complete detection of risk
patients is not successful and therefore a risk stratification is carried
out only to an unsatisfactory extent. One further object on which the
invention is based therefore lies in developing a method for the risk
stratification of cardiac insufficiency, which renders possible an
improved detection of risk patients.

[0010]The object is attained through a method for the diagnosis and/or
risk stratification of cardiac insufficiency, wherein a determination of
the marker neurophysin or a fragment or partial peptide thereof is
carried out on a patient to be examined (hereinafter referred to as the
method according to the invention).

[0011]Within the scope of this invention "cardiac insufficiency" is
understood to mean an acute or chronic inability of the heart to supply
tissue with sufficient blood and as a result thereof with sufficient
oxygen, in order to ensure tissue metabolism at rest or under stress.
Clinically, a cardiac insufficiency is present when typical symptoms
(dyspnea, fatigue, liquid retention) exist which are based in origin on a
cardiac functional disorder in terms of a systolic or diastolic
functional disorder. Chronic cardiac insufficiency (CHF) is likewise
covered according to the invention (Kardiologie compact, edited by
Chrisian Mewis, Reimer Riessen and Ioakim Spyridopouolos, 2nd
unamended edition, Thieme 2006). The causes of a cardiac insufficiency
can be: cardiac valve defect (e.g., as the late symptom of rheumatic
fever), myocarditis (inflammation of the myocardium), cardiac
arrhythmias, cardiac infarction together with excessively high blood
pressure (hypertonia) and/or arteriosclerosis (calcification) of the
coronary vessels (coronary heart disease). Furthermore, the following are
covered according to the invention: hypertensive heart disease with
(congestive) cardiac insufficiency, hypertensive heart disease and kidney
disease with (congestive) cardiac insufficiency, primary dextrocardiac
insufficiency, secondary dextrocardiac insufficiency, left ventricular
insufficiency without symptoms (NYHA stage I), left ventricular
insufficiency with symptoms with greater stress (NYHA stage II), left
ventricular insufficiency with symptoms with slight stress (NYHA stage
III), left ventricular insufficiency with symptoms at rest (NYHA stage
IV) and cardiogenic shock.

[0012]The method according to the invention therefore likewise covers the
above-referenced indications. Furthermore, all of the cited indications
are described, e.g., in Pschyrembel, De Gruyter, Berlin 2004.

[0013]According to the invention, the term "risk stratification" covers
discovering patients, in particular emergency patients and risk patients,
with the worse prognosis for the purpose of more intensive diagnosis and
therapy/treatment of cardiac insufficiency with the objective of
rendering possible the most favorable possible progress. A risk
stratification according to the invention consequently allows an
effective treatment method, which are given with respect to a cardiac
insufficiency.

[0014]Particularly advantageously, in particular in cases of emergency
and/or intensive care medicine, a reliable diagnosis can be made by means
of the method according to the invention. The method according to the
invention renders possible clinical decisions that lead to a rapid
therapeutic success. Clinical decisions of this type likewise include
further treatment by means of pharmaceuticals for the treatment or
therapy of cardiac insufficiency, such as ACE inhibitors, AT1
antagonists: blockers of the angiotensin II receptor (subtype I), beta
blockers bisoprolol, carvedilol, metoprolol and nebivolol, vasopressin
receptor antagonists, aldosterone antagonists from NYHA stage III,
calcium sensitizers (Levosimendan).

[0015]In a further preferred embodiment the method according to the
invention therefore relates to the therapeutic control of a cardiac
insufficiency.

[0016]The invention therefore likewise relates to a method for the risk
stratification of patients, in particular for the stratification of
patients for clinical decisions, preferably in time-critical intensive
medicine or emergency medicine and for the hospitalization of patients

[0017]In a further preferred embodiment of the method according to the
invention the diagnosis is carried out for prognosis, for the
differential diagnostic early detection and detection, for the assessment
of the degree of severity and for the assessment of the course of a
cardiac insufficiency accompanying therapy.

[0018]In a further embodiment of the method according to the invention
body fluid, in particular blood, optionally whole blood or serum, is
taken from the patient to be examined and the diagnosis is carried out in
vitro/ex vivo, i.e., outside the human or animal body. The diagnosis can
be carried out based on the determination of the marker neurophysin and
the quantity thereof present in at least one patient sample.

[0019]Within the scope of this invention "neurophysin" (or neurophysin II)
(fragment: AS32-124 of the preprovasopressin; see FIG. 2) is understood
to be a polypeptide/protein containing free 93 amino acid (93AS: SEQ ID
no. 1: AMSDLELRQC LPCGPGGKGR CFGPSICCAD ELGCFVGTAE ALRCQEENYL PSPCQSGQKA
CGSGGRCAAF GVCCNDESCV TEPECREGFH RRA) or fragments or partial peptides
thereof. Furthermore, this polypeptide according to the invention can
have posttranslational modifications, such as glycolization,
lip(o)idization or derivatizations. Neurophysin is surprisingly stable in
plasma.

[0020]In a further embodiment the determination of neurophysin can
additionally be made with further markers, namely preferably those that
already indicate a cardiac insufficiency and permit a synergistic effect
of marker combinations containing neurophysin in the method according to
the invention.

[0021]The invention therefore relates to such an embodiment of the method
according to the invention, wherein the determination is additionally
carried out on a patient to be examined with at least one further marker
selected from the group of inflammatory markers, cardiovascular markers,
neurohormonal markers or ischemic markers.

[0022]According to the invention, the inflammatory marker can be selected
from at least one marker from the group of C-reactive protein (CRP),
cytokinin, such as, for example, TNF-alpha, interleukins, such as, for
example, IL-6, procalcitonin (1-116, 3-116) and adhesins, such as VCAM or
ICAM, and the cardiovascular marker from at least one marker from the
group creatincinase, myeloperoxidase, copeptin, myoglobin, natriuretic
protein, in particular ANP (or ANF), proANP, NT-proANP, BNP, proBNP,
NT-proBNP or respectively a partial sequence thereof, cardial troponin,
CRP. Furthermore, these are likewise understood to include (pro)hormones
regulating the circulation, in particular like pro-gastrin-releasing
peptide (proGRP), pro-endothelin (proEnd), pro-leptin,
pro-neuropeptide-Y, pro-somatostatin, pro-neuropeptide-YY,
pro-opiomelanocortin, pro-adrenomedullin (proADM), copeptin or
respectively a partial sequence thereof.

[0023]The ischemic marker can be selected from at least one marker from
the group troponin and T, CK-MB. Furthermore, the neurohormonal marker
can be at least one natriuretic protein, in particular ANP (or ANF),
proANP, NT-proANP, BNP, proBNP, NT-proBNP or respectively a partial
sequence thereof.

[0025]In a further embodiment of the invention the method according to the
invention can be carried out by means of parallel or simultaneous
determinations of the markers (e.g., multititration plates with 96 and
more cavities), wherein the determinations are carried out on at least
one patient sample.

[0026]Furthermore, the method according to the invention and the
determinations thereof can be carried out on an automatic analyzer, in
particular by means of a Kryptor (http://www.kryptor.net/).

[0027]In another embodiment, the method according to the invention and the
determinations thereof can be carried out by means of a rapid test (e.g.,
a lateral-flow test), whether in single-parameter or multiple-parameter
determination.

[0028]Furthermore, the invention relates to the use of neurophysin or a
fragment or partial peptide thereof and optionally further markers, as
listed above, for the diagnosis and/or risk stratification of cardiac
insufficiency.

[0029]Another object is the provision of a corresponding diagnostic device
or the use thereof for carrying out the method according to the
invention.

[0030]Within the scope of this invention, a diagnostic device in
particular an array or assay (e.g., immunoassay, ELISA, etc.) is
understood to be in the broadest sense a device for carrying out the
method according to the invention.

[0031]The invention furthermore relates to a kit for the diagnosis or risk
stratification of cardiac insufficiency, containing analytical reagents
for determining the marker neurophysin or a fragment or partial peptide
thereof and optionally the markers listed above. Analytical reagents of
this type comprise, e.g., antibodies, antibody fluorescence, etc.

[0032]The following examples and figures are used to explain the invention
in more detail, but without restricting the invention to these examples
and figures.

EXAMPLES AND FIGURES

[0033]A blood sample was taken from patients who reported to the emergency
room of a hospital with the indicating symptom of respiratory distress
during the initial examination. EDTA plasma obtained through
centrifugation was aliquoted and stored at -80° C. until the
measurement of neurophysin. According to earlier descriptions (Pullan
(supra)) a radioimmunoassay was developed for neurophysin:
neurohypophyseal neurophysin was isolated and quantified. Rabbits were
immunized therewith and thus high-titered anti-neurophysin antisera
obtained. For the immunoassay the highest-titered antiserum was used in a
concentration of 1:100,000. Purified neurophysin was radio-iodized with
the chloramines T method and used as a tracer in the assay. Dilutions of
purified neurophysin in normal horse serum were used as standards. The
assay was carried out as follows: 50 μl sample or standard was mixed
with 100 μl tracer (12,000 dpm per determination) and 100 μl
diluted anti-neurophysin antiserum and incubated for 24 hours at
4° C. 100 mM sodium phosphate, pH 7.5, 0.1% BSA was used as a
buffer. Antibody-bound tracer was separated from free tracer in that 60%
ethanol was added and then centrifuged for 15 minutes at 4° C. and
5,000 g. The supernatant was discarded and the radioactivity remaining in
the pellet was determined. The evaluation was carried out with the aid of
Multicalc software. The assay had an analytical detection limit of 22
pg/ml and a measuring range up to 400 pg/ml. Plasma samples from
different patients, as explained below, were measured with the assay.
Samples with measured values>400 pg/ml were measured in suitable
dilutions, so that measured values within the measuring range were
obtained.

[0035]Furthermore, blood was taken from healthy individuals without known
disease and EDTA plasma was obtained through centrifugation.

[0036]Clinical Utility

[0037]Normal Range

[0038]Neurophysin concentrations were determined in samples from healthy
control subjects (n=20). The median was 70.3 pg/ml, the lowest measured
value was 8.8, the highest 220 pg/ml, the 95% percentiles 36.5 or 135
pg/ml.

[0039]Cardiac Insufficiency/Degree of Severity

[0040]Neurophysin concentrations were measured in patients with chronic or
acute decompensated cardiac insufficiency. Neurophysin concentrations
were associated with the degree of severity of the cardiac insufficiency:
the average values of the neurophysin concentrations in the four NYHA
categories of degrees of severity I-IV were: 171.4, 243.4, 346.9 and
918.1 pg/ml respectively (see FIG. 1).

[0041]Chronic Cardiac Insufficiency/Diagnosis

[0042]Neurophysin values were determined from a group of 258 patients with
chronic cardiac insufficiency and 200 healthy controls. The receiver
operator characteristics analysis yielded an AUC of 0.89. With a cut-off
value of 213 pg/ml, a sensitivity of 48% resulted with a specificity of
98%. With a cut-off value of 136.1 pg/ml, a sensitivity of 68.2% resulted
with a specificity of 95%.

[0043]Chronic Cardiac Insufficiency/Prognosis

[0044]Neurophysin values were determined from a group of 258 patients with
chronic cardiac insufficiency. The patients were observed over an average
period of 360 days. Within this period 80 patients died, 178 survived.
The best cut-off value (defined as the greatest product from sensitivity
and specificity) for the prognosis of mortality was determined through
receiver operator characteristics analysis: 247 pg/ml. With this cut-off
value the sensitivity of the prognosis was 59.5%, the specificity was
65.2%. The likelihood ratio with a cut-off value of 247 pg/ml was 1.7
(see table below).

TABLE-US-00001
<247 pg/ml >247 pg/ml
Survived 116 62
Died 32 47

[0045]Acute Cardiac Insufficiency/Diagnosis

[0046]Neurophysin values were determined from a group of 125 patients with
acute respiratory distress. 69 patients out of the 125 patients had
cardiac insufficiency. The receiver operator characteristics analysis for
the differential diagnosis of the cardiac insufficiency yielded an AUC of
0.61. With a cut-off value of 4940 pg/ml, a sensitivity of 6.6% resulted
with a specificity of 98%. With a cut-off value of 3000 pg/ml, a
sensitivity of 11.7% resulted with a specificity of 95%.

[0047]Acute Cardiac Insufficiency/Prognosis

[0048]Neurophysin values were determined from a group of 69 patients with
acute decompensated cardiac insufficiency. The patients were observed
over a period of 360 days. Within this period 21 patients died, 48
survived. The best cut-off value (defined as the greatest product of
sensitivity and specificity) was determined for the prognosis of
mortality through receiver operator characteristics analysis: 885 pg/ml.
With this cut-off value the sensitivity of the prognosis was 57.1%, the
specificity was 75%. The likelihood ratio with a cut-off value of 885
pg/ml was 2.3 (see table below).